Gu D , Sater AK , Ji H , Cho K , Clark M , Stratton SA , Barton MC , Lu Q , McCrea PD .

CA-16672 NCI NIH HHS , R01 CA111891 NCI NIH HHS , R01 GM52112 NIGMS NIH HHS , R01 GM052112 NIGMS NIH HHS

	Fig. 3. Spatial expression of Xenopus -catenin. (A) As viewed in animal versus vegetal regions, whole-mount in situ RNA hybridization detects -catenin mRNA signals in the ectoderm regions of blastula (subpanels A-C) and gastrula (subpanel D) embryos. At neurulation (subpanel E), the anterior and dorsal neural regions displayed the most apparent signals. Embryos at tadpole stages (subpanel F) showed a distinctive staining pattern in tissues of neural derivation such as brain, eye vesicle, ear vesicle, branchial arches (higher magnification in subpanel G) and spinal cord as well as somites (higher magnification in subpanel H). Subpanels I-L are cross-section views of paraffin-fixed embryos from corresponding stages. Sense probe hybridization was processed in parallel as negative controls (subpanels M-O). (B) RT-PCR analyses detect -catenin transcripts in all adult Xenopus tissues examined, with stronger expression in brain, nerve, muscle and skin. (C) Immunoblotting using an N-terminus-directed antibody detected three -catenin isoforms migrating at approximately 160, 130 and 100 kDa. The 130 and 100 kDa isoforms are ubiquitously present, whereas the 160 kDa appears to be brain specific. An antibody directed against the -catenin C-terminus reacts with the 160 kDa and 130 kDa isoforms in brain.
	Figure 2. enopus δ-catenin temporal expression profiles. (A) Schematic diagram of δ-catenin alternative splicing events, and the PCR primers used to resolve them. The nucleotide lengths of PCR products are indicated in parentheses. (B) RT-PCR analyses indicate δ-catenin transcripts are deposited maternally and expressed throughout early embryonic stages. Both long (a,b,c) and short (a′,b′,c′) splicing variants were detected, with b′ and c′ having increased expression following neurulation. (C) Immunoblotting confirms δ-catenin protein expression throughout Xenopus embryogenesis. Antibodies directed against Xenopus amino acids 83-521 (N-terminal domain) recognize a δ-catenin isoform migrating at approximately 100 kDa; antibodies directed against Xenopus amino acids 1297-1314 (C-terminal domain) react mainly with a 100 kDa doublet, with reactivity additionally appearing at 130 kDa (marked with an asterisk) and 160 kDa (not shown, but see Fig. 3C). The 130 and 160 kDa bands are most evident following immunoprecipitation (results not shown).
	Figure 4. Xenopus δ-catenin associates with classical cadherins and displays a significant non-membrane-associated fraction. Endogenous δ-catenin complexes were immunoprecipitated using a C-terminus-directed antibody (1297-1314), and immunoblotted using antibodies direct against cadherins. Positive co-immunoprecipitation results suggest an association of δ-catenin with C-cadherin in gastrulating embryos (A), and with E-cadherin (B) and N-cadherin (C) in neurulation stage embryos. Immunoglobulin heavy chain (IgG H.C.) bands are included to reflect the specific versus negative control antibody input. (D) Crude membrane fractionations of gastrula embryos followed by immunoblot analyses indicate the predominant localization (∼70%) of endogenous δ-catenin within non-plasma-membrane pools. As expected, GAPDH is almost exclusively evident in the non-plasma-membrane pools. C-cadherin predominantly resides within the plasma-membrane pool (65%), with the remaining fraction likely to reflect associations with non-sedimenting vesicular stores, endoplasmic reticulum or Golgi.
	Figure 5. Antisense morpholino depletion of endogenous δ-catenin results in developmental defects. (A) Schematic diagram of the morpholino-based strategy, including the relative positions of PCR diagnostic primers. MO 6 targets δ-catenin RNA at the splice junction between intron 5 and exon 6, whereas MO 9 targets the junction between intron 8 and exon 9. MO 6 produces exon 6 elimination, with predicted codon frame-shift and early polypeptide termination. In addition to the normal transcript, RT-PCR confirmed (via DNA sequencing) the expected alteration in pre-RNA splicing (using oligos F1+R2; marked with an asterisk). Likewise, MO 9 results in alternative splicing, exon elimination and translational termination (PCR product marked with a single apostrophe was confirmed to be skipping of exon 9 and part of exon 8. An additional partial exon 10 deletion is indicated with a double apostrophe). δ-catenin transcription and mRNA stability did not appear to be significantly altered (oligos D and C, see also Fig. 2A), with histone H4 serving as an internal control. (B) Immunoblotting of gastrula stage embryo extracts confirmed the reduction of δ-catenin protein following morpholino injection. Non-specific bands (labeled with asterisks) and β-actin act as loading controls. Numbers indicate relative band intensities normalized to the uninjected control. (C) δ-catenin knockdown results in developmental phenotypes, including significant delays in blastopore closures and gastrulation defects (upper right panel). Although most embryos outwardly appear to recover from these effects and complete blastopore closure, the majority of MO-9-injected embryos were developmentally arrested during early tailbud or tadpole stages and subsequently died. For surviving embryos, abnormalities were again outwardly evident, particularly at tadpole stages, including shortened anterior-posterior axes, smaller craniofacial skeletons and eyes, malformed gut and edema (lower panel). Control embryos injected with standard morpholino displayed no obvious phenotypes (upper left and middle panel). P-values indicate statistical significance.
	Figure 6. Fig. 6. δ-catenin-knockdown phenotypes are rescued by overexpression of either δ-catenin or p120-catenin. (A) The specificity of δ-catenin-depletion phenotypes were verified through rescue with select constructs. Exogenous and titrated FL (full-length) δ-catenin (see also supplementary material Fig. 4A,B) as well as M434 δ-catenin (initiated with the fourth potential translation start site, see also Fig. 1) largely rescued blastopore closure defects arising from endogenous δ-catenin depletion. Titrated levels of p120-catenin, but not β-catenin, also reproducibly displayed significant rescuing activity. (B) Co-injection of δ-catenin and p120-catenin morpholino, each at subphenotypic doses, produces enhanced phenotypic effects.(C) Use of a depletion-rescue strategy suggests that the PDZ-binding motif of δ-catenin is dispensable for the rescuing capacity of δ-catenin in blastopore closure. By contrast, the δ-catenin N-terminus or armadillo domain in isolation failed to rescue δ-catenin depletion. (D) Schematic presentation of various rescue constructs with a summary of their rescuing effects (asterisk indicates data from Fig. 7D). For all panels P-values indicate statistical significances.
	Figure 7. δ-catenin depletion leads to reduced cadherin functions. (A) Immunoblotting shows that δ-catenin depletion reproducibly leads to reduced C-, E- and N-cadherin levels, whereas p120-catenin and β-catenin levels are not significantly altered.(B) Calcium-dependant adhesive functions were decreased in δ-catenin-depleted naive ectoderm cells. Following calcium removal from ectoderm explants, note the larger cell aggregates remaining after control MO injection. (C) Using an in vitro assay with the extracellular domain of E-cadherin tethered to a solid substrate (chamber glass), cadherin-mediated adhesion is decreased in naive ectoderm cells depleted of δ-catenin. By contrast, a similar assay that uses tethered fibronectin, did not resolve changes in cell attachment (presumably integrin mediated). (D) A titrated dose of exogenous C-cadherin significantly rescues blastopore closure defects induced by depletion of endogenous δ-catenin. By contrast, a δ-catenin mutant construct lacking armadillo repeats 1-5, and failing to co-immunoprecipitate with C-cadherin (supplementary material Fig. 5A) showed minimal rescuing effects. For all panels P-values indicate statistical significance.

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